| Abstract [eng] |
Due to various factors, the production of some recombinant proteins in the yeast S. cerevisiae is inefficient. Two-dimensional electrophoresis (2DE) and mass spectrometry (MS)-based proteomic analysis is a powerful tool which may help reveal the reasons for the inefficient synthesis of these proteins. To find the most suitable 2DE method for yeast proteome analysis, we compared two 2DE methods to study the inefficient translocation of measles virus hemagglutinin (MeH) protein to the endoplasmic reticulum (ER). We found that in a wide pH range, non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE is the preferred method for yeast proteome analysis, over the immobilized pH gradient (IPG)-based 2DE. Also, we showed that the application of heat shock to high-density cell cultures increases the efficiency of MeH translocation and the amount of glycosylated protein by ~3 times. After analyzing the proteomes of cells grown under conditions of more efficient translocation using 2DE methods, we identified 15 protein targets possibly related to improved MeH translocation. We have restored the gel solution and carrier ampholyte composition for NEPHGE-2DE that is suitable for use in place of commercially no longer available solutions. Furthermore, we performed a comparative, quantitative, NEPHGE-2DE and LC-MSE - based proteomic analysis of human calreticulin protein (CALR)-secreting and control yeast cells. Our results show, that the efficient secretion of CALR in the yeast S. cerevisiae does not burden the yeast secretory machinery and does not induce any apparent cellular stress. These results show that NEPHGE-based 2DE combined with MS is a suitable method for proteomic analysis of the recombinant protein-synthesizing yeast S. cerevisiae. |
